ABSTRACT AD is characterized by a progressive loss of memories over time. This has been hypothesized to be due to a loss of long-term potentiation (LTP) and other learning and memory mechanisms in neurons affected during Alzheimer?s Disease (AD) pathogenesis, a result observed both in rodent AD models as well as humans1,2. Much like AD patients lose the ability to recall former memories, AD model rodents also show deficits in memory recall3,4. This is presumably due to a destabilization of existing memories, though this has not been directly examined. If we could stabilize plasticity against destabilization that occurs during AD pathogenesis, we could prolong the persistence of adaptive memories, thus substantially reducing the symptomatic burden of AD patients and their caregivers. As LTP is thought to be a molecular substrate of memories, loss of LTP should be shortly followed by a loss of associated behavioral memories. Indeed, evidence suggests that this is the case; we find that deterioration of LTP in the hippocampus in the 5xFAD rodent model starts at roughly 4-6 months of age, followed closely by the loss of previously established hippocampal memories3,4. In our funded NIH Director?s Innovator Award (DP2AG067666), we proposed to develop a suite of molecular methods to modulate plasticity in a spatiotemporally-defined fashion. These approaches are based on the elucidation of the molecular mechanism of the small peptide ZIP, which we have found to work through macropinocytosis, as ZIP?s behavioral effects could be blocked by prior administration of amiloride. Importantly, we showed that amiloride not only blocks ZIP- induced destabilization of LTP, but also natural processes that remove AMPAR receptors, such as are required for long-term depression (LTD)-induced behavioral extinction. This raises the possibility that our methods for plasticity modulation, including amiloride administration and molecularly-defined variants, could be used as a therapeutic strategy to prevent destabilization of LTP that occurs during AD pathogenesis. This in turn could slow the development of AD-associated cognitive and behavioral deficits. In this administrative supplement application, we will test the ability of our approaches to slow the AD-associated loss of contextual and auditory fear conditioning memories, as well as conditioned place preference memories. Contextual fear memories can be disrupted by modulating LTP in the hippocampus5,6, and we show in our preliminary data that auditory fear conditioning memories are stored in the basolateral amygdala (BLA), and conditioned place preference memories are stored in the nucleus accumbens (NAc). We show that injection of ZIP or the small cationic peptide TAT into the BLA or NAc eliminates the associated memory, and the effects of ZIP/TAT were blocked by prior amiloride infusion. Here we will test the maintenance of these three memories in the 5xFAD AD model mice at two different ages following 1 month of either chronic amiloride administration or knockdown of Thorase, both of which effectively prevent AMPAR endocytosis. Time permitting, we will examine memory deficits in a different model of AD, P301S/E47. Aim 1: Test effect of chronic amiloride on memory maintenance in AD model mice. Our preliminary data suggest that long-term amiloride infusion my prevent or slow normal memory loss associated with AD. In these experiments we will have mice learn both an auditory and contextual fear memory as well as a drug-paired conditioned place memory, and chronically infuse amiloride into either the hippocampus, BLA, or NAc after the association has been learned. Memories will be established at 6 or 9 months, and amiloride will be infused starting 1 day later. Recall for each memory will be tested 1 month later. Aim 2: Test effects of conditional Thorase knockdown on memory maintenance in AD model mice. Mice with deletions of the Thorase gene have deficiencies in plasticity, as AMPARs can no longer be properly removed from synapses8,9. Although this impairs learning in mice constitutively lacking the gene, preventing AMPAR removal would effectively ?lock? a memory in after it has been established, which should have a similar effect as chronic amiloride administration. Therefore, after having mice undergo fear conditioning and conditioned place preference training, Thorase mRNA will be reduced selectively in the hippocampus, BLA, or NAc through small hairpin RNA-mediated knockdown, delivered by adeno-associated viruses. Behaviors will be tested as in Aim 1. By identifying methods to prolong the persistence of long-term memories in AD models, these studies could lay the foundation for development of future AD therapeutics. Future studies using targeted approaches in defined cell types in different AD models would help to elucidate the role of plasticity destabilization in defined cell types in the progression of AD pathogenesis. We are actively pursuing such an approach by using rabies virus-based methods to identify key neuronal populations beyond the hippocampus and cortex that play important roles in the behavioral deficits observed in rodent AD models, including 5xFAD. When we identify these populations, we will use the approaches proposed in this application to test the effects of plasticity stabilization on the development of AD-associated cognitive and behavioral deficits.